In our clinical trials we are interested to test novel treatment approaches or to gain new insights into mechanisms of action or mechanisms of drug resistance to agents used in the treatment of CLL. We conduct a clinical trial using lenalidomide (protocol 07-H-0104), a novel drug with promising phase II results. The mechanism of action and predictors of treatment outcome for this drug in CLL have not been defined. In a first step, we aim to measure the gene expression changes in the leukemic cells in blood and lymph nodes during treatment. A second trial is devoted to test the hypothesis whether frequent low dose administration of rituximab could overcome resistance mechanism against this agent in CLL (06-H-0228). In a collaborative study with the NCI lymphoma team, we obtain CLL cells from patients who undergo therapy with two of the most active drugs in CLL therapy;rituximab and fludarabine. Patients donate blood every day during the first 6 treatment days and we analyze the changes in gene expression in the leukemic cells due to the therapy. Mechanisms how the monoclonal anti-CD20 antibody rituximab (R) depletes B-cells include antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. In vitro studies have suggested that R induced pro-apoptotic signals contribute to clinical efficacy and may sensitize malignant cells to chemotherapy. To investigate the effect of R on tumor biology in vivo, we characterized the molecular changes in chronic lymphocytic leukemia cells of 12 treatment nave patients during the first R infusion. We first determined whether R treatment affects gene expression in the circulating leukemic cells. CLL cells obtained before and at 6 and 24 hours after the start of R were purified by CD19+ selection and gene expression was measured on Affymetrix HU133A 2.0 arrays. We identified about 80 genes up-regulated in response to R infusion, many of which are known to be regulated by interferon (IFN) and to have pro-apoptotic function. We found that IFN gamma was consistently upregulated in the serum within the first 6 hours of treatment. Considering the long half-life of the monoclonal antibody, we were surprised to see that both cytokine serum levels and gene expression changes almost completely subsided by 24 hours. We therefore assessed CD20 protein levels in circulating leukemia cells at 6 and 24hours from the start of treatment by Western blotting. Total CD20 levels were markedly decreased already at 6 hours and by 24 hours almost all CD20 had been lost consistent with a process previously described as shaving, during which R bound CD20 is pulled of the cell surface. Our data show that infusion of R induced a characteristic gene expression signature in CLL cells that is dominated by IFN response genes, many of which have well characterized pro-apoptotic functions. We conclude that signaling for apoptosis is less of a direct effect of R and more due to a complex immune response to the R coated CLL cells. Modified administration schedules delivering repeat pulses of the pro-apoptotic signals while reducing loss of CD20 expression hold promise for improved efficacy of R and should be explored. To investigate the nurturing and protective role of stroma cells, we analyze gene expression patterns in CLL cells f that we obtain from peripheral blood, bone marrow and lymph nodes. Our hypothesis is that the leukemic cells receive essential proliferation and survival signals in the bone marrow and/or lymph node. We have analyzed 8 matched pairs of bone marrow and blood derived CLL cells. Consistent with our hypothesis we found that CLL cells from the bone marrow showed a higher expression of genes associated with proliferation as well as a couple hundred genes that may relate to the specific signals induced in the leukemic cells by specific signals provided by the bone marrow microenvironment. We are extending this study now to lymphnode samples and are following up on candidate molecules that could play a role in stimulating CLL cell survival. In addition, we are establishing models in vitro where we can show that the survival of CLL cells is extended by co-culture with stroma cells. From these studies we hope to identify which signaling pathways are essential for leukemic cell survival and which therefore could be good targets of new therapies.